Methods of generating corneal cells and cell populations comprising same

ABSTRACT

A method of generating a population of corneal epithelial cells is disclosed. The method comprises culturing human pluripotent stem cells in corneal fibroblast-conditioned medium on a solid surface comprising an extracellular matrix component thereby generating the population of corneal epithelial cells. Isolated cell populations and corneal tissues are also disclosed, as well as uses thereof.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof generating corneal cells from pluripotent stem cells and cellpopulations comprising same.

The cornea is a unique, transparent structure that covers the iris,pupil, and anterior chamber, providing most of the eye's optical power.Together with the lens, the cornea refracts light and, as a result, aidsin focusing. The cornea contributes more to the total refraction thanthe lens does, but, whereas the curvature of the lens can be adjusted to“tune” focus, the curvature of the cornea is fixed. The cornea has noblood vessels, its nourishment is obtained via diffusion from the tearfluid, the aqueous humor, and neurotrophins supplied by nerve fibersthat innervate it. Thus, for example, disturbances in circulation ofthese fluids or inflammatory processes play a large role in thepathogenesis of corneal abnormalities.

The cornea is composed mostly of dense connective tissue. However, thecollagen fibers are arranged in a parallel pattern, allowing light wavesto constructively interfere, thus letting light pass through relativelyuninhibited.

The corneal tissue is arranged in five basic layers: epithelium,Bowman's layer, stroma, Descemet's membrane and endothelium, each havinga separate function. The epithelium is the outermost layer of thecornea, comprising about 10% of the tissue's thickness. The epitheliumfunctions primarily to: (1) block passage of foreign materials, such asdust, water, and bacteria, into the eye and other layers of the cornea;and (2) provide a smooth surface that absorbs oxygen and cell nutrientsfrom tears, then distributes these nutrients to the rest of the cornea.The corneal epithelium is maintained by stein cells (SCs) located at theperiphery of the cornea, in a region known as the limbus. The cornealepithelium itself is devoid of its own stem cells. Limbal fibroblastsform the major cellular component of the limbal stroma upon which theLSCs reside, and they produce specific cytokines that promote cornealepithelial wound healing by the LSCS.

The changes associated with aging of the cornea include increasedopacity, increased anterior surface curvatures, and possibly changes inrefractive index distribution. Various refractive eye surgery techniqueschange the shape of the cornea in order to reduce the need forcorrective lenses or otherwise improve the refractive state of the eye.In many techniques, reshaping of the cornea is performed byphotoablation using an eximer laser.

If the corneal stroma develops visually significant opacity,irregularity, or edema, a cadaveric donor cornea can be transplanted.

Limbal auto-grafts have been applied to patients with a relatively highdegree of success. In severe cases such as total limbal stem celldeficiency, allo-grafts may be obtained from patient's relatives or frompost mortem donors. However, limbal tissue is highly immunogenic and therate of graft rejection exceeds 35%, 5 years post transplantation. Thismethod is further limited due to a shortage of donors. Further, livingdonors are at risk of developing limbal stem cell deficiency. To reducethis risk, a smaller limbal tissue may be grafted from donor andexpanded ex vivo prior to transplantation. This technology does notallow treating patient immediately following injury, since it requiresthe cultivation of the cells for several weeks prior to grafting.Moreover, allogenic limbal stem cell transplantations to a patient areeventually rejected.

Synthetic corneas also exist (keratoprotheses), however, these aretypically plastic inserts or may be composed of biocompatible syntheticmaterials that encourage tissue in-growth into the synthetic cornea,thereby promoting biointegration. Alternatively, orthokeratology offersthe use of specialized hard or rigid gas-permeable contact lenses totransiently reshape the cornea in order to improve the refractive stateof the eye or reduce the need for eyeglasses and contact lenses.

Homma et al. [Invest Ophthalmol Vis Sci. 2004 December; 45(12):4320-6]teach corneal epithelial differentiation of murine embryonic bodies oncollagen IV-coated dishes.

Sajjad et al. [Stem Cells. 2007 May; 25(5):1145-55] teach generation ofcorneal epithelial-like cells by seeding human embryonic stem cells oncollagen IV-coated dishes in the presence of medium that was conditionedby limbal fibroblasts. The resulting corneal epithelial cells werecontaminated with skin cells.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a population of cornealepithelial cells comprising culturing human pluripotent stem cells incorneal fibroblast-conditioned medium on a solid surface comprising anextracellular matrix component thereby generating the population ofcorneal epithelial cells.

According to some embodiments of the invention, the cornealfibroblast-conditioned medium comprises bone morphogenetic protein-4(BMP-4).

According to some embodiments of the invention, the human pluripotentstem cells comprise human embryonic stem cells (hESCs) or human inducedpluripotent stem cells (hlPSCs).

According to some embodiments of the invention, the cornealfibroblast-conditioned medium comprises at least one agent selected fromthe group consisting of insulin, hydrocortisone and epidermal growthfactor (EGF). According to some embodiments of the invention, thecorneal fibroblast-conditioned medium comprises insulin, hydrocortisoneand epidermal growth factor (EGF).

According to some embodiments of the invention, the extracellular matrixcomponent is selected from the group consisting of collagen IV, laminin,fibronectin and Matrigel^(RTM.)

According to some embodiments of the invention, the method furthercomprises analyzing the population of corneal epithelial cells for atleast one marker selected from the group consisting of keratin 3 (K3),keratin 12 (K12), paired box gene 6 (pax6), keratin 18 (K18) andConnexin 43.

According to some embodiments of the invention, the cornealfibroblast-conditioned medium is devoid of limbal fibroblasts.

According to some embodiments of the invention, there is provided anisolated population of human corneal epithelial cells generatedaccording to the method of the present invention.

According to some embodiments of the invention, the isolated populationof corneal epithelial cells does not comprise skin cells.

According to some embodiments of the invention, at least 70% of thecells of the population co-express K3 and Pax6.

According to some embodiments of the invention, less than 10% of thecells of the population express nanog and Oct4.

According to some embodiments of the invention, the isolated populationof corneal epithelial cells is for use in treating an eye disorder.

According to some embodiments of the invention, there is provided amethod of treating an eye disorder in a subject in need thereof, themethod comprising transplanting to the subject a therapeuticallyeffective amount of corneal epithelial cells generated according to themethod of the present invention, thereby treating the eye disorder inthe subject.

According to some embodiments of the invention, there is provided qapharmaceutical composition comprising the isolated population of cornealepithelial cells of the present invention and a pharmaceuticallyacceptable carrier.

According to some embodiments of the invention, there is provided amethod of generating corneal tissue, the method comprising:

-   -   (a) dissociating the isolated population of human corneal        epithelial cells of the present invention to generate a        population of dissociated human corneal epithelial cells; and    -   (b) culturing the dissociated human corneal epithelial cells on        a scaffold under conditions that generate corneal tissue.

According to some embodiments of the invention, the scaffold comprisesMatrigel® and collagen I.

According to some embodiments of the invention, the scaffold comprises ahuman amniotic membrane.

According to some embodiments of the invention, there is provided anisolated human corneal tissue generated according to the method of thepresent invention.

According to some embodiments of the invention, there is provided ahuman corneal tissue for use in treating an eye disorder.

According to some embodiments of the invention, there is provided amethod of treating an eye disorder in a subject in need thereof, themethod comprising transplanting to the subject a therapeuticallyeffective amount of corneal tissue generated according to the method ofthe present invention, thereby treating the eye disorder in the subject.

According to some embodiments of the invention, there is provided apharmaceutical composition comprising the isolated corneal tissue of thepresent invention and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a method of screening for an agent which enhancesdifferentiation towards a corneal epithelial lineage, the methodcomprising:

(a) culturing human pluripotent stem cells in cornealfibroblast-conditioned medium on a solid surface comprising anextracellular matrix component in a presence of said agent; and

(b) analyzing a differentiation status of said human pluripotent stemcells, wherein an increase in differentiation compared to adifferentiation in an absence of said agent is indicative of an agentwhich enhances differentiation towards a corneal epithelial lineage.

According to some embodiments of the invention, the pluripotent stemcells comprise iPS cells.

According to some embodiments of the invention, the iPS cells arederived from healthy patients.

According to some embodiments of the invention, the iPS cells arederived from diseased patients.

According to some embodiments of the invention, the medium furthercomprises BMP-4.

According to some embodiments of the invention, the diseased patientsare ectrodactyly—ectodermal dysplasia—cleft syndrome (EEC) patients.

According to some embodiments of the invention, the analyzing iseffected by analyzing a morphology of said pluripotent stem cells.

According to some embodiments of the invention, the analyzing iseffected by analyzing an expression of a corneal cell marker in saidpluripotent stem cells.

According to some embodiments of the invention, the corneal cell markeris Pax6 or K3/K12.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIGS. 1A-G illustrate corneal commitment of human embryonic stem (huES)cells. (A). Morphology of a confluent human ES cell population after 10days of corneal differentiation. (B-E) Kinetic analysis of corneal andpluripotent markers at different days of corneal differentiation of huEScells. Cells, RNA and protein extracts were collected at different timesof commitment for qRT-PCR (B), Western blot (C), immunofluorescence (D)and FACS (E) analysis, showing efficient production of cornea-specificmarker (pax-6, K12, K3, connexin 43) and disappearance ofpluripotent-specific markers (oct-4 and nanog). (F and G) Specificity ofthe corneal differentiation of huES cells. (F) Corneal-specific DNAconstruct carrying the fluorescent GFP gene under the K12 promoter wastransfected into several cell lines and human ES cells during cornealcommitment and quantification was done by FACS analysis of GFP-positivecells. The corneal K12 promoter was active in human corneal epithelialcontrol cells (HCE) and in huES committed cells at D6 and D12 and not inundifferentiated ES cells or non corneal epithelial cell lines (Hela,HaCaT). (G) Differentiated epidermal-specific markers K1, K10 andinvolucrin were not expressed by undifferentiated (D0) or huES cellscommitted into corneal differentiation (D6, D12 and D17), as compared tonormal huma, keratinocytes (NHK), used as positive control.

FIGS. 2A-C illustrate production of corneal epithelium from human EScells. Corneal fibroblasts were embedded in collagen type 1 gel toproduce a corneal stroma equivalent (A). After a week, huES-derivedcorneal cells were seeded on the top of the gel for one week to allowproliferation. Then, the culture was lifted up at the air-liquidinterface to induce stratification (B). (C) Immunofluoresence analysisdemonstrated that the resulting corneal epithelia were positive for K3(red) and pax-6 (green; dapi in blue).

FIGS. 3A-C are photographs illustrating production of corneal epithelialtissue using amniotic membrane as a scaffold.

FIGS. 4A-C are graphs illustrating differentiation of iPSC cells intocorneal epithelial lineage. iPSC colonies were seeded on collagenIV-coated dishes in the presence of epithelial-medium that wasconditioned by corneal fibroblasts. (A). RNA preparations obtained inthe indicated days of corneal differentiation were subjected to realtime PCR analysis of the indicated genes. The mRNA expression levelswere normalized to GAPDH. Data represents the relative fold change ofeach transcript as compared to its levels as recorded inundifferentiated hES cells. (B). Test of BMP-4. (C). Corneal commitmentof hair follicle-derived iPSCs (HF2-iPS), fibroblast-derived iPSCs (DF1and DF2-iPS) and human ES cells quantified by real time qRT-PCR analysisfor K18, pax6, DNp63, K14 and K3 genes.

FIGS. 5A-B are photographs illustrating corneal commitment of iPS celllines.

FIGS. 6A-B are photographs and graphs illustrating impaired cornealdifferentiation of EEC-iPS cells.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof generating corneal cells from pluripotent stem cells and cellpopulations comprising same.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

When a cornea becomes cloudy due to disease or injury, light cannotpenetrate the eye to reach the light-sensitive retina. Poor vision orblindness may result. Corneal transplant surgery involves the removal ofthe central portion of the cloudy cornea and replacing it with a clearcornea, usually donated through an eye bank. This technique is hamperedby a scarcity of donors and graft rejection.

Limbal auto-grafts have been applied to patients with disorders of thecornea with a relatively high degree of success. In severe cases such astotal limbal stem cell deficiency, allo-grafts may be obtained frompatient's relatives or from post mortem donors. However, limbal tissueis highly immunogenic and the rate of graft rejection exceeds 35%, 5years post transplantation. This method is further limited due to ashortage of donors. Further, living donors are at risk of developinglimbal stem cell deficiency. To reduce this risk, a smaller limbaltissue may be grafted from donor and expanded ex vivo prior totransplantation. This technology does not allow treating patientimmediately following injury, since it requires the cultivation of thecells for several weeks prior to grafting. Moreover, allogenic limbalstem cell transplantations to a patient are eventually rejected.

In order to overcome these problems, the present inventors devised atechnique whereby they generated corneal epithelial cell populationsfrom pluripotent stem cells. The differentiation protocol relies on theuse of corneal fibroblast-conditioned medium.

As illustrated in FIGS. 1A-G, the generated corneal epithelial cellpopulation was homogeneous, the cells expressing K3, K12, pax6 andConnexin43. Furthermore, the present inventors noted that the cellpopulation was devoid of cells expressing epidermal markers such as K1,K10 and Involucrin (see FIG. 1G).

The present inventors further showed that 3D culture of such cellsresulted in the generation of corneal tissue equivalents (FIG. 2). Thisprocedure allows the production of ready to use tissues for cornealtransplantation, thereby resolving the need of donors.

Thus, according to one aspect of the present invention there is provideda method of generating a population of corneal epithelial cellscomprising culturing human pluripotent stem cells in cornealfibroblast-conditioned medium on a solid surface comprising anextracellular matrix component thereby generating the population ofcorneal epithelial cells.

The method of the present invention is initially effected by obtainingpluripotent stem cells and culturing them.

As used herein, the phrase “stem cells” refers to cells which arecapable of remaining in an undifferentiated state (e.g., pluripotent ormultipotent stem cells) for extended periods of time in culture untilinduced to differentiate into other cell types having a particular,specialized function (e.g., fully differentiated cells). The phrase“pluripotent stem cells” encompasses embryonic stem cells (ESCs) andinduced pluripotent stem cells (iPS. The stem cells are typicallymammalian pluripotent cells, such as for example human pluripotent stemcells.

The phrase “embryonic stern cells” refers to embryonic cells which arecapable of differentiating into cells of all three embryonic germ layers(i.e., endoderm, ectoderm and mesoderm), or remaining in anundifferentiated state. The phrase “embryonic stem cells” may comprisecells which are obtained from the embryonic tissue formed aftergestation (e.g., blastocyst) before implantation of the embryo (i.e., apre-implantation blastocyst), extended blastocyst cells (EBCs) which areobtained from a post-implantation/pre-gastrulation stage blastocyst (seeWO2006/040763) and embryonic germ (EG) cells which are obtained from thegenital tissue of a fetus any time during gestation, preferably before10 weeks of gestation.

Induced pluripotent stem cells (iPS; embryonic-like stem cells), arecells obtained by de-differentiation of adult somatic cells which areendowed with pluripotency (i.e., being capable of differentiating intothe three embryonic germ cell layers, i.e., endoderm, ectoderm andmesoderm). According to some embodiments of the invention, such cellsare obtained from a differentiated tissue (e.g., a somatic tissue suchas skin or hair) and undergo de-differentiation by genetic manipulationwhich re-program the cell to acquire embryonic stem cellscharacteristics. According to some embodiments of the invention, theinduced pluripotent stem cells are formed by inducing the expression ofOct-4, Sox2, Kfl4 and c-Myc in a somatic stem cell.

The embryonic stem cells of the present invention can be obtained usingwell-known cell-culture methods. For example, human embryonic stem cellscan be isolated from human blastocysts. Human blastocysts are typicallyobtained from human in vivo preimplantation embryos or from in vitrofertilized (IVF) embryos. Alternatively, a single cell human embryo canhe expanded to the blastocyst stage. For the isolation of human ES cellsthe zona pellucida is removed from the blastocyst and the inner cellmass (ICM) is isolated by immunosurgery, in which the trophectodermcells are lysed and removed from the intact ICM by gentle pipetting. TheICM is then plated in a tissue culture flask containing the appropriatemedium which enables its outgrowth. Following 9 to 15 days, the ICMderived outgrowth is dissociated into clumps either by a mechanicaldissociation or by an enzymatic degradation and the cells are thenre-plated on a fresh tissue culture medium. Colonies demonstratingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and re-plated. Resulting ES cellsare then routinely split every 4-7 days. For further details on methodsof preparation human ES cells see Thomson et al., [U.S. Pat. No.5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998;Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can alsobe used with this aspect of the present invention. Human ES cells can bepurchased from the NIH human embryonic stem cells registry(www.escr.nih.gov). Non-limiting examples of commercially availableembryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92,CY10, 1E03 and 1E32.

In addition, ES cells can be obtained from other species as well,including mouse (Mills and Bradley, 2001), golden hamster [Doetschman etal., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, DevBiol. 163: 288-92] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8;Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], severaldomestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl.43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova etal., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesusmonkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci U S A.92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of atleast nine days post fertilization at a stage prior to gastrulation.Prior to culturing the blastocyst, the zona pellucida is digested [forexample by Tyrode's acidic solution (Sigma Aldrich, St Louis, Mo., USA)]so as to expose the inner cell mass. The blastocysts are then culturedas whole embryos for at least nine and no more than fourteen days postfertilization (i.e., prior to the gastrulation event) in vitro usingstandard embryonic stem cell culturing methods.

EG cells are prepared from the primordial germ cells obtained fromfetuses of about 8-11 weeks of gestation (in the case of a human fetus)using laboratory techniques known to anyone skilled in the arts. Thegenital ridges are dissociated and cut into small chunks which arethereafter disaggregated into cells by mechanical dissociation. The EGcells are then grown in tissue culture flasks with the appropriatemedium. The cells are cultured with daily replacement of medium until acell morphology consistent with EG cells is observed, typically after7-30 days or 1-4 passages. For additional details on methods ofpreparation human EG cells see Shamblott et al., [Proc. Natl. Acad. Sci.USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.

Induced pluripotent stem cells (iPS) (embryonic-like stem cells) can begenerated from somatic cells by genetic manipulation of somatic cells,e.g., by retroviral transduction of somatic cells such as fibroblasts,hepatocytes, gastric epithelial cells with transcription factors such asOct-3/4, Sox2, c-Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007,1(1):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells fromAdult Mouse Liver and Stomach Cells. Science. 2008 Feb 14. (Epub aheadof print); IH Park, Zhao R, West JA, et al. Reprogramming of humansomatic cells to pluripotency with defined factors. Nature2008;451:141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction ofpluripotent stem cells from adult human fibroblasts by defined factors.Cell 2007;131:861-872]. Other embryonic-like stem cells can be generatedby nuclear transfer to oocytes, fusion with embryonic stem cells ornuclear transfer into zygotes if the recipient cells are arrested inmitosis.

It will be appreciated that undifferentiated stem cells are of adistinct morphology, which is clearly distinguishable fromdifferentiated cells of embryo or adult origin by the skilled in theart. Typically, undifferentiated stem cells have highnuclear/cytoplasmic ratios, prominent nucleoli and compact colonyformation with poorly discernable cell junctions. Additional features ofundifferentiated stem cells are further described hereinunder.

Currently practiced ES culturing methods are mainly based on the use offeeder cell layers which secrete factors needed for stem cellproliferation, while at the same time, inhibit their differentiation.Feeder cell free systems have also been used in ES cell culturing, suchsystems utilize matrices supplemented with serum, cytokines and growthfactors as a replacement for the feeder cell layer.

In order to induce differentiation of the pluripotent stem cells to thecorneal epithelial lineage, the pluripotent stem cells are cultured incorneal fibroblast-conditioned medium.

Corneal fibroblasts may be obtained from cadavers or living donors.According to one embodiment, the corneal fibroblasts are obtained fromhumans. Typically corneal fibroblasts are isolated by incubation of thecornea with a dispersing agent (e.g. Dispase II, typsin or collagenasefor about 1-18 hours at 37° C.). The epithelial sheet may then beremoved with forceps. Care should be taken to avoid contamination of thecorneal sample with limbal tissue, such that the cells used to generatethe conditioned medium are not contaminated with limbal fibroblasts.

Conditioned medium is the growth medium of a monolayer cell culture(i.e., feeder cells) present following a certain culturing period. Theconditioned medium includes growth factors and cytokines secreted by themonolayer cells in the culture.

In order to produce corneal fibroblast conditioned medium, the isolatedfibroblasts may initially be expanded by culturing in a suitable medium(e.g. dulbeco's modified eagle medium (DMEM) supplemented with 10% newborn calf serum). When the fibroblast cell population reaches about80%-100% density (see FIG. 2A), cell proliferation is stopped, e.g. byincubation in mitomycin C (8 μg/ml) for about three hours. Themitomycinized cells are then incubated in a suitable growth medium.

Conditioned medium is collected from the corneal fibroblasts formingmonolayers in culture.

The growth medium can be any medium suitable for culturing the cornealfibroblasts. The growth medium can be supplemented with nutritionalfactors, such as amino acids, (e.g., L-glutamine), anti-oxidants (e.g.,beta-mercaptoethanol) and growth factors, which benefit cornealfibroblast cell growth.

According to one embodiment bone morphogenetic protein-4 (BMP-4) may beadded during the culture period. The BMP-4 may be added for the entirelength of the culturing period or at a particular stage of the culturingperiod (e.g. for the first three days of the culturing period).

An exemplary concentration of BMP-4 is between about 0.1-10 nM, morepreferably between 0.1 - 5 nM, more preferably between about 0.1 and 1nM (for example 0.5 nM).

According to one embodiment, the growth medium comprises at least one ofthe following agents—insulin (e.g. 5 μg/ml), hydrocortisone (e.g. 0.5μg/ml) and EGF e.g. (10 ng/ml).

According to another embodiment the growth medium comprises insulin,hydrocortisone and EGF.

An exemplary medium is epithelial medium ((DMEM 60%, HamF12 30%, FCII10%, insulin 5 μg/ml, hydrocortisone 0.5 EGF 10 ng/ml, 0.2 mM Adenine,10 nM Cholera toxin).

The epithelial fibroblast cells are cultured in the growth medium forsufficient time to allow adequate accumulation of secreted factors tosupport stem cell differentiation towards the corneal epithelial celllineage. Typically, the medium is conditioned by culturing for 4 hoursat 37° C. to 24 hours.

However, the culturing period can be scaled by assessing the effect ofthe conditioned medium on stem cell differentiation.

Selection of culture apparatus for conditioning the medium is based onthe scale and purpose of the conditioned medium. Large-scale productionpreferably involves the use of dedicated devices. Continuous cellculture systems are reviewed in Furey (2000) Genetic Eng. News 20:10.

Following accumulation of adequate factors in the medium, growth medium(i.e., conditioned medium) is separated from the corneal fibroblasts andcollected. It will be appreciated that the corneal fibroblasts can beused repeatedly to condition further batches of medium over additionalculture periods, provided that the cells retain their ability tocondition the medium.

According to one embodiment, the medium is collected every day andreplaced by fresh media for up to 10 days.

Preferably, the conditioned medium is sterilized (e.g., filtration usinga 20 μM filter) prior to use. The conditioned medium of some embodimentsof the invention may be applied directly on stem cells or extracted toconcentrate the effective factor such as by salt filtration. For futureuse, conditioned medium is preferably stored frozen at −80° C.

Culturing in corneal fibroblast-conditioned medium is typically effectedon a solid surface which comprises an extracellular matrix component.Examples of extracellular matrix components include but are not limitedto collagen IV, laminin, fibronectin and Matrigel®.

Methods of coating culture dishes with extraceullar matrix componentsare well known in the art and further described in the Examples sectionherein below.

The pluripotent stem cells are cultured under suitable conditions toallow differentiation. According to one embodiment, the cells arecultured for about 5 days, 7 days or about 10 days.

The differentiation status of the cells may be determined by analyzingmarker expression thereof. Thus for example the following markers may beanalyzed: cytokeratin 3 (K3), cytokeratin 12 (K12), paired box gene 6(pax6), cytokeratin 18 (K18) and Connexin 43. Expression of the abovedescribed markers in at least 50%, at least 60%, at least 70%, at least80%, at least 90% of the generated cell population may be used as anindication that the cell population is differentiated towards thecorneal epithelial lineage.

According to a particular embodiment, the population of cells isselected wherein at least 70%, 80% or 90% of the cells of the populationco-express K3 and Pax6.

Alternatively, or additionally, the differentiation status of the cellsmay be determined by analyzing markers of undifferentiated stem cells,such as Oct4 or nanog. Expression of the above described markers in lessthan 40%, less than 30%, less than 20%, less than 10%, less than 5% ofthe generated cell population may be used as an indication that the cellpopulation comprises differentiated cells.

Preferably the generated population of cells does not comprise skincells. Accordingly, the population of cells may be analyzed for skincell markers (e.g. K1, K10 and Involucrin) and only cell populationswhich do not express such markers (e.g. less than 10% of the cellsexpress or less than 5% of the cells express) are selected.

Methods for analyzing cells for particular markers are well known in theart and include for Example Western Blot Analysis, flow cytometry andreverse transcriptase PCR analysis (RT-PCR). Immunohistochemistrytechniques may be used to ascertain whether a particular cellco-expresses two or more markers.

Antibodies for K3, K12, Pax6, K18, K1, K10, involucrin and connexin 43are commercially available from Companies such as Santa CruzBiotechnology and Chemicon.

Exemplary primer sequences which may be used to analyze thedifferentiation status of the cells are provided herein below.

Pax-6 (forward): (SEQ ID NO: 1) gCTTggTggTgTCTTTgTCA Pax-6 (reverse):(SEQ ID NO: 2) TCACACAACCgTTggATACC connexin 43 (forward):(SEQ ID NO: 3) gCTgAgCCCTgCCAAAgAC connexin 43 (reverse): (SEQ ID NO: 4)gAggAgCAgCCATTgAAATAA

The differentiation status of the cells may also be determined byanalyzing promoter activity for a particular gene known to be expressedin differentiated cells. Example 1 of the Examples section herein belowdetails how the activity of the k12 promoter (a corneal specificpromoter) may be analyzed in the differentiated cell populations.

The above described differentiation protocol may be used to screen foragents which enhance differentiation towards a corneal epitheliallineage.

Thus, according to another aspect of the present invention, there isprovided a method of screening for an agent which enhancesdifferentiation towards a corneal epithelial lineage, the methodcomprising:

(a) culturing human pluripotent stem cells in cornealfibroblast-conditioned medium on a solid surface comprising anextracellular matrix component in a presence of said agent; and

(b) analyzing a differentiation status of the human pluripotent stemcells, wherein an increase in differentiation compared to adifferentiation in an absence of the agent is indicative of an agentwhich enhances differentiation towards a corneal epithelial lineage.

Analyzing the differentiation status of the cells has been describedherein above.

The pluripotent stem cells may be embryonic stem cells or inducedpluripotent stem cells (iPS cells). The iPS cells may be derived fromhealthy subjects or patients who have a disease which affects cornealepithelial cells - for example, ectrodactyly—ectodermal dysplasia—cleftsyndrome (EEC) patients. The present inventors have shown that iPS cellsderived from such patients do not undergo differentiation to cornealepithelial cells to the same extent as iPS cells derived from healthysubjects. Thus, the present protocol may be used to screen for a drugthat enhances corneal epithelial cell differentiation for the treatmentof a particular disease.

Examples of agents that may be screened include polypeptide agents,polynucleotide agents, small molecule agents and other chemicals.

The isolated cell populations generated according to the method of thisaspect of the present invention may be used for various applicationsincluding transplantation thereof for the treatment of diseases ordisorders of the cornea, as further described herein below. In addition,the isolated cell populations may be used as a cellular model for drugdiscovery and cell toxicity tests. The reproducibility of identicalcells from the same source will allow comparative and standardizesstudies, which is an important criteria for industrials of cosmetologyand pharmacology.

Alternatively, the isolated cell populations (e.g. ones that have beeninitially differentiated for 1-2 weeks in the corneal fibroblastconditioned medium) may be further differentiated on a 3D scaffold togenerate corneal tissue.

According to one embodiment, the corneal tissue is stratified.

According to another embodiment, the corneal tissue comprises at least 3layers of corneal epithelial tissue.

According to another embodiment the corneal tissue is devoid of squamouscells.

A method of generating corneal tissue on a scaffold is described inExample 2 of the Examples section below and illustrated in FIGS. 3A-I.

According to one embodiment the 3D scaffold comprises a gel composed ofMatrigel^(RTM) and collagen I.

According to another embodiment, the scaffold comprises a human amnioticmembrane.

Such techniques are described in Koizumi et al., InvestigativeOphthalmology & Visual Science, August 2000, Vol. 41, No. 9; Gaggioli etal., Nature cell biology volume 9, number 12, 2007 and Larouch et al.,Chapter 15, Stem Cells in Regenerative Medicine: Methods and Protocols,vol. 482, 2009), the contents of each being incorporated by reference.

Typically, the cell populations are differentiated on the scaffold forabout 1-2 weeks to induce differentiation of corneal tissue.

As mentioned, the isolated corneal epithelial cell populations (and thecorneal tissue differentiated therefrom) may be used to treat diseasesand disorders of the cornea.

Disorders affecting the cornea include, but are not limited to,allergies, conjunctivitis, corneal infections, dry eye, Fuchs'dystrophy, herpes zoster, iridocorneal endothelial syndrome,keratoconus, lattice dystrophy, map-dot-fingerprint dystrophy, ocularherpes, Stevens-Johnson syndrome, pterygium, keratitis, corneal ulcer,corneal abrasion, snow blindness, arc eye, Thygeson's superficialpuncate keratopathy, and keratpconjunctivitis sicca.

In corneal transplant surgery, the surgeon typically removes the centralportion of the diseased or injured cornea and replaces it with a clearcornea. The new cornea or corneal cells are placed in the opening and issutured to the eye (see, e.g., Rapuano et al. Anterior Segment, TheRequisites (Requisites in Ophthalmology), 1999, Mosby, Inc.,Philadelphia, Pa.). Typically about 1-2×10⁶ cells/eye are transplantedto the damaged cornea.

In one embodiment, a method for replacement of a cornea of an eye usingthe synthetic cornea of the present invention includes surgicallyexcising the cornea from the eye, inserting the synthetic cornea intothe area of the removed cornea, and allowing the synthetic cornea tointerface with tissue underlying the excision to anchor the syntheticcornea to the eye.

In a related aspect, the method includes separating a portion of theouter surface of a cornea thereby forming a corneal flap and a cornealbed, the corneal flap having an anterior surface and a posteriorsurface, the corneal bed having a shaped anterior surface, implantingthe synthetic cornea on the corneal bed, the cornea having an anteriorsurface and a posterior surface, and replacing the portion of the corneathat was separated.

Careful histological and immunohistochemical analysis should beperformed at different times following grafting by using specificmarkers. The number of goblet cells on the corneal surface should beevaluated with impression cytology to evaluate the presence of limbaldeficiency. The rejection index, the mean survival time, and therejection rates may be calculated for each group.

As mentioned hereinabove, the corneal cells or tissue of the presentinvention can be derived from either autologous sources (inducedpluripotent stem cells) or from allogeneic sources such as embryonicstem cells. Since non-autologous cells are likely to induce an immunereaction when administered to the body several approaches have beendeveloped to reduce the likelihood of rejection of non-autologous cells.For example, prior to transplantation, the histo-compatibility of thesubject may be tested such that only histo-compatible corneal grafts maybe transplanted.

Other approaches include either suppressing the recipient immune systemor encapsulating the non-autologous cells in immunoisolating,semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu MZ, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesThechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 μm (CanapleL. et al., Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002;13:783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironments for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE.sup.R), etanercept, TNF.alpha. blockers, abiological agent that targets an inflammatory cytokine, andNon-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDsinclude, but are not limited to acetyl salicylic acid, choline magnesiumsalicylate, diflunisal, magnesium salicylate, salsalate, sodiumsalicylate, diclofenac, etodolac, fenoprofen, flurbiprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

If appropriate, the patient can be further treated with pharmaceuticalagents or bioactives that facilitate the survival and function of thetransplanted cells. These agents may include, for example, insulin,members of the TGF-beta family, including TGF-beta1, 2, and 3, bonemorphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13),fibroblast growth factors-1 and -2, platelet-derived growth factor-AA,and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growthdifferentiation factor (GDF-5, -6, -7, -8, -10, -15), vascularendothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin,among others. Other pharmaceutical compounds can include, for example,nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1 and 2mimetibody, Exendin-4, retinoic acid, parathyroid hormone, MAPKinhibitors, such as, for example, compounds disclosed in U.S. PublishedApplication 2004/0209901 and U.S. Published Application 2004/0132729.

The cells or tissue of the present invention may be transplanted to ahuman subject per se, or in a pharmaceutical composition where it ismixed with suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the cell populations described herein with other chemicalcomponents such as physiologically suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (corneal cells) effective to prevent, alleviate orameliorate symptoms of a disorder.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated from animalmodels to achieve a desired concentration or titer. Such information canbe used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures inexperimental animals. The data obtained from these animal studies can beused in formulating a range of dosage for use in human. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provide cellnumbers sufficient to induce repair or improve sight (minimal effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Differentiation of Human Embryonic Stem Cells to Corneal Cells

I. Preparation of corneal fibroblasts conditioned epithelial medium: Toprepare conditioned media, primary corneal fibroblasts were extractedfrom human cornea that is not suitable for transplantation by incubationof the cornea with Dispase II (2 mg/ml) overnight at 37° C. Theepithelial sheet was removed and the fibroblasts were cultured inculture dishes in dulbeco's modified eagle medium (DMEM) supplementedwith 10% new born calf serum. When the fibroblast cell populationreached 80%-100% density (see FIG. 2A), cell proliferation was stoppedby incubating in mitomycin C (8 μg/ml) for three hours. To obtainconditioned media, the mitomycinized cells were incubated withepithelial media ((DMEM 60%, HamF12 30%, FCII 10%, insulin 5 μg/ml,hydrocortisone 0.5 EGF 10 ng/ml, 0.2 mM Adenine, 10 nM Cholera toxin).The medium was collected every day and replaced by fresh media for up to10 days. The medium was filtered and stored at −20° C. until use. II.Coating of culture dishes: Culture dishes were incubated with collagenIV (0.5 mg/ml) or matrigel (0.25 mg/ml) at 37° C. for 4 hours and thenwashed extensively with phosphate buffered saline.

III. Differentiation of pluripotent stem cells into corneal epitheliallineage: Pluripotent stem cell cells were incubated with collagenase (2mg/ml) for 1 hour and then colonies were seeded on collagen IV ormatrigel-coated dishes. Medium was replaced every second day by freshcorneal fibroblast conditioned epithelial media for about two weeks.

Real-time PCR analysis: RNA extractions of cells during different timepoint of corneal differentiation were analyzed by real-time PCR analysisof K18, K12, pax6, GAPDH.

Western blot analysis: we used anti K3/K12 Ab, anti pax6 Ab, anti K18Ab, anti Connexin 43 Ab, anti Nanog Ab, anti Oct4 Ab.

FACS analysis: FACS analysis was performed using anti K18 Ab, antiK3/K12 Ab and anti pax6 Ab.

Transfection of cells with GFP under a K12 promoter: Embryonic stemcells-derived corneal cells (at Day 10 of differentiation), and cellsthat were used as positive (Human corneal epithelial (HCE) immortalizedcell line) or negative (HaCaT, Hela, undifferentiated ES cells) controlswere transfected with a GFP-encoding plasmid that is under K12-cornealspecific promoter, using Fugene reagent according the manufacturerinstructions. After 48 hours, the percentage of cells with GFPfluorescence was examined by FACS analysis.

Results

The morphology of the differentiated cells appeared relativelyhomogenous at day 9, as shown in FIG. 1A. Corneal differentiation wasevaluated by quantitative real time polymerase chain reaction (qPCR) ofcorneal epithelial markers (FIG. 1B). Elevated levels of the ectodermalmarker K18 (and K8, not shown), and of pax6, a key factor in eye andcorneal development, were recorded in early culture days. The mRNAlevels of corneal specific cytokeratins, namely K12 and K3 wereincreased at late culture days (FIG. 1B).

Western blot analysis demonstrated that the protein levels of knowncorneal-epithelial markers (namely, pax6, K3, K12 and Connexin43)increased gradually at late differentiation stages while embryonic stemcell markers (Nanog and Oct4) became undetectable within 6-8 days (FIG.1C), suggesting that this protocol induces efficient cornealdifferentiation of ES cells.

The co-expression of K18 and pax6 hallmarks the ‘lens placode’, astructure that emerges at day 9.5 of mouse embryogenesis (E9.5), fromwhich the corneal epithelium is derived. At later embryonic stages, thecorneal epithelium maintains pax6-expression and begins to express K12and K3. Notably, the majority of the cells at day 4 of ESdifferentiation expressed K18 or co-expressed K18 and pax6 (FIG. 1D,left panel), while, at day 10 of ES differentiation, the majority ofcells co-expressed K3 and pax6 (FIG. 1D), suggesting that ES cellsdifferentiate into corneal precursors in a manner that reminiscent ofcorneal embryogenesis.

To further assess corneal epithelial fate, differentiated cells weresubjected to FACS analysis of K18, pax6 and K3. As shown in FIG. 1E,most of the cells expressed K18 and pax6 at day 6, while the vastmajority of the cells expressed K3 and pax6 (>90%) at day 12.Furthermore, the encoding sequence for green fluorescent protein (GFP)was cloned under the corneal-specific promoter of K12 gene, which is themost specific amongst corneal markers. The specificity of this constructto corneal epithelial cells was confirmed by the transfection of variousnon-corneal cell lines as negative controls (Hela, HaCaT, ES) or of ahuman corneal epithelial cell line (HCE) as positive control (FIG. 1F).While non-corneal cells did not express any significant amount of GFP,78% of HCE and 71% of ES-derived corneal epithelial-like (ES-EC) cellsdisplayed a strong K12-promoter activity (FIG. 1F). Finally, noepidermal differentiation markers (namely K1, K10 and Involucrin, seeFIG. 1G) were detected, suggesting that highly enriched population ofES-CE cells were obtained that was not contaminated with epidermal-likecells.

Example 2 Generation of Corneal Tissue from Embryonic Stem Cells

Materials and Methods

After 1-2 weeks of differentiation of pluripotent stem cells on collagenIV or matrigel in the presence of conditioned media, cells werecollected by dispase or trypsin and reseeded onto a corneal stromaequivalent gels in a 3D organotypic reconstitution assay: primary humancorneal fibroblast cells were embedded in gels that were composed ofmatrigel and collagen I, and hES-derived corneal cells were seeded ontop of these gels in epithelial media. Corneal stratification wasinduced by air-liquid interphase as illustrated in FIGS. 2A-C andfurther exemplified in Gaggioli et al., Nature cell biology volume 9,number 12, 2007. Then air liquid interface was induced by reducing mediainside the inserts and cells were allowed to stratify for 1-2 weeks,while medium was replaced every day. Alternatively, differentiated cellswere seeded on human amniotic membrane and lifted from the culture (asdescribed for instance in Koizumi et al., Investigative Ophthalmology &Visual Science, August 2000, Vol. 41, No. 9).

Example 3 Differentiation of Human Induced Pluripotent Stem (iPS) Cellsto Corneal Cells

I. Preparation of corneal fibroblasts conditioned epithelial medium: asdescribed in Example 1.

II. Coating of culture dishes: as described in Example 1.

III. Differentiation of iPS cells into corneal epithelial lineage: iPSderived from human hair follicle cells or dermal fibroblasts were seededon collagen IV or matrigel-coated dishes. Medium was replaced everysecond day by fresh corneal fibroblast conditioned epithelial media forabout two weeks. On days 3, 8 and 14, BMP-4 was added to the medium.

Real-time PCR analysis: RNA extractions of cells during different timepoint of corneal differentiation were analyzed by real-time PCR analysisof K3, K14, K12, K18, pax6 and DNp63.

FACS analysis: FACS analysis was performed using anti K14 Ab, antiK3/K12 Ab and anti pax6 Ab.

Results

Addition of BMP-4 during the first three days significantly enhancedcorneal differentiation, as illustrated by increased expression ofDNp63, K14 and K12 (FIG. 4A). This effect was inhibited by LDN, aspecific antagonist of BMP-4. To follow corneal commitment, messengerRNA levels of corneal epithelial lineage genes were recorded atdifferent time points in the course of iPSCs differentiation by qPCR(FIG. 4B). Elevation in ectodermal marker K18 (and K8, not shown)appeared already within 2-4 culture days (FIG. 4B), along with theexpression of pax6, an early marker of neuroectodermal cell fate and akey regulator of eye development. Early epithelial commitment wasdetected at days 6-8 by the expression of limbal markers, p63 and K14,while markers of mature corneal epithelium (K3, K12) and Connexin 43(not shown) appeared within 10-14 days (FIG. 4B). Simultaneousexpression of K18 and pax6 at early stage recapitulated the in vivoco-expression in the lens placode at E9.5, was followed by co-expressionof pax6 and K3 which are hallmark of mature corneal cells. Therobustness of corneal epithelial-like cell production was evaluated atday 12 by FACS analysis. The vast majority of the cells expressed K3(>90%) while 20% of the cells remained K14-positive cells. Finally,although epidermal marker K10 increased at the mRNA levels, no K10protein could be detected (not shown). Similar results were obtainedwith iPSCs derived from human fibroblasts or huESCs (FIG. 4C).

Example 4 Differentiation of Diseased iPS Cells Towards a Corneal Fate

EEC patients suffer from visual morbidity due to impaired corneaassociated with limbal stem cell deficiency. iPSC lines were induced tocorneal fate as described in Example 3. As illustrated by real-timeqRT-PCR analysis, human iPSC lines underwent sequential differentiationinto ectodermal precursors (K8/K18+/Pax6⁺) at day 4, limbal(K14/K5/Pax6⁺/p63⁺) at day 8 and corneal epithelial (Pax-6⁺/K3/K12⁺)cells at day 14 (FIG. 5A). Remarkably, at day 14, most of the cellsbecame corneal epithelial cells, as detected by immunofluorescentstaining (FIG. 5B) and FACS analysis (data not shown). Since EECpatients suffer from limbal stem cell deficiency, iPS^(EEC) cells werechallenged for their ability to undergo proper corneal epithelialcommitment as compared to the iPSC^(ctl). Immunofluorescent staininganalysis was performed at day 10 with antibodies raised against K18,E-cadherin and K14 (FIG. 6A). Similar production of ectodermalprogenitors (K18⁺/E-cadherin⁺) was observed at day 10 for iPSC^(ctl),iPSC^(204W) and iPSC^(304W). However, the absence of K14 and K3 stainingrevealed the inability of iPSC^(EEC) to undergo further commitment forthe production of limbal cells and corneal cells, respectively, ascompared to iPSC^(ctl). In parallel, gene expression of known p63-targetgenes was evaluated at day 10 of commitment by real-time qRT-PCR (FIG.6B). Most of the genes were less expressed in mutated cells as comparedto control cells.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1-31. (canceled)
 32. A method of treating an eye disorder in a subjectin need thereof, the method comprising transplanting to the subject atherapeutically effective amount of human corneal epithelial cellsgenerated according to a method of generating a population of humancorneal epithelial cells which comprises culturing human pluripotentstem cells in corneal fibroblast-conditioned medium on a solid surfacecomprising an extracellular matrix component thereby generating thepopulation of human corneal epithelial cells, wherein said cornealfibroblast-conditioned medium is produced from human corneal fibroblastswhich are not contaminated with limbal fibroblasts and comprisesinsulin, hydrocortisone and epidermal growth factor (EGF).
 33. Themethod according to claim 32, wherein said cornealfibroblast-conditioned medium further comprises bone morphogeneticprotein-4 (BMP-4).
 34. The method according to claim 32, wherein saidextracellular matrix component is selected from the group consisting ofcollagen IV, laminin, fibronectin and Matrigel®.
 35. The methodaccording to claim 32, wherein said population of human cornealepithelial cells does not comprise skin cells.
 36. The method accordingto claim 32, wherein at least 70% of the cells of said population ofhuman corneal epithelial cells co-express K3 and Pax6.
 37. The methodaccording to claim 32, wherein wherein less than 10% of the cells ofsaid population of human corneal epithelial cells express nanog andOct4.
 38. A method of treating an eye disorder in a subject in needthereof, the method comprising transplanting to the subject atherapeutically effective amount of human corneal tissue generatedaccording to a method of generating human corneal tissue whichcomprises: (a) culturing human pluripotent stem cells in cornealfibroblast-conditioned medium on a solid surface comprising anextracellular matrix component, thereby generating a population of humancorneal epithelial cells, wherein said corneal fibroblast-conditionedmedium is produced from human corneal fibroblasts which are notcontaminated with limbal fibroblasts and comprises insulin,hydrocortisone and epidermal growth factor (EGF); (b) dissociating thegenerated population of human corneal epithelial cells to generate apopulation of dissociated human corneal epithelial cells; and (c)culturing said dissociated human corneal epithelial cells on a scaffoldunder conditions that generate human corneal tissue.
 39. The methodaccording to claim 38, wherein said corneal fibroblast-conditionedmedium further comprises bone morphogenetic protein-4 (BMP-4).
 41. Themethod according to claim 38, wherein said extracellular matrixcomponent is selected from the group consisting of collagen IV, laminin,fibronectin and Matrigel®.
 42. The method according to claim 38, whereinsaid population of human corneal epithelial cells does not comprise skincells.
 43. The method according to claim 38, wherein at least 70% of thecells of said population of human corneal epithelial cells co-express K3and Pax6.
 44. The method according to claim 38, wherein wherein lessthan 10% of the cells of said population of human corneal epithelialcells express nanog and Oct4.
 45. A method of screening for an agentwhich enhances differentiation towards a human corneal epitheliallineage, the method comprising: (a) culturing human pluripotent stemcells in corneal fibroblast-conditioned medium, wherein said cornealfibroblast-conditioned medium is produced from human corneal fibroblastswhich are not contaminated with limbal fibroblasts and comprisesinsulin, hydrocortisone and epidermal growth factor (EGF), on a solidsurface comprising an extracellular matrix component in a presence ofsaid agent; and (b) analyzing a differentiation status of said humanpluripotent stem cells, wherein an increase in differentiation comparedto a differentiation in an absence of said agent is indicative of anagent which enhances differentiation towards a human corneal epitheliallineage.
 46. The method of claim 45, wherein said pluripotent stem cellscomprise iPS cells.
 47. The method of claim 45, wherein said iPS cellsare derived from healthy patients.
 48. The method of claim 45, whereinsaid iPS cells are derived from diseased patients.
 49. The method ofclaim 45, wherein said corneal fibroblast-conditioned medium furthercomprises bone morphogenetic protein-4 (BMP-4).